The effect on the function of the transcriptional activator NtrC from Klebsiella pneumoniae of mutations in the DNA-recognition helix

Abstract

We have constructed mutations in what we predict to be the DNA-recognition helix of Klebsiella pneumoniae NtrC, which regulates transcription from promoters under global nitrogen control. Mutations which disrupt the helix lead to complete loss of function. All point mutants tested were able to activate transcription from the sigma 54-dependent glnA promoter, but only those retaining some ability to recognise NtrC binding sites, as evidenced by their ability to repress the ntrB promoter and the upstream glnA promoter, were able to activate the nifL promoter. One mutant, which contained an amino acid substitution in the turn of the DNA-binding motif as well as in the recognition helix, suppressed mutations in the NtrC binding sites upstream from the nifL promoter, but only if both sites bore equivalent transitions. This confirms that the DNA-binding motif for this class of transcriptional activator has been correctly identified and suggests that binding of NtrC can be cooperative.